PROJECT SUMMARY/ABSTRACT Monitoring local concentrations of neurochemicals in specific parts of the brain in vivo is critical for correlating neural circuit functionality to behavior as long-range neuromodulation can significantly alter information processing. Current methods for detecting neuromodulators have limited temporal and/or spatial resolution, limited sensitivity and/or are prohibitively invasive. Our long-term goal is to develop a highly sensitive multiplexed platform technology for simultaneous detection of local concentrations of various neurochemicals in the brain that can be time-synchronized with recordings of neural activity, optogenetic stimulation, and behavior. The overarching objective is to develop a silicon-based microfluidic nanodialysis probe able to collect in-vivo a series of isolated sub-nanoliter (nL) volume analytes from the brain extracellular fluid (ECF) with less than 100 ?m spatial, less than 1 s temporal resolution, and with limits of detection approaching basal levels in a time- sequential manner synchronized with animal behavior. In three specific aims, we will develop and test the probe in awake behaving animals: (Aim1) Develop a library of microfluidic devices and systems integrated on silicon nanodialysis probes with an overall cross-section minimized to avoid excessive tissue damage. (Aim 2) The nanodialysis probe will be co-integrated with the mass-spectrometry (MS) which will characterize the complex chemical mosaic within the samples to provide for time-synchronized detection of multiple neuromodulators simultaneously. (Aim 3) The new instrument will be thoroughly tested for tissue damage, chemical and temporal sensitivity and, finally, in neurobiology experiments where the electrophysiological neural activity will be detected in close proximity to the time-synchronized nanodialysis probe sampling. The experiments will be conducted in-vivo in awake and behaving animals and the spatial and temporal transients of neurochemical concentrations will be correlated with the neural activity and behavior. This contribution is significant since the instrumental platform will allow monitoring concentration gradients of various neuromodulators and drugs from precise brain locations time-synchronized with recordings of neural activity and behavior that should enable advances in fundamental systems neuroscience as well as accelerate development of new treatments for neurological diseases. The proposed research is innovative because we will develop highly sensitive nanodialysis probes on silicon platform that allows us to attain unprecedented temporal and spatial resolution. The developed versatile silicon platform enables future integration of nanodialysis functionality with electrophysiological recordings and optogenetic stimulation on a single neural probe and will significantly facilitate broad dissemination of this manufacturable technology.